Olfactomedin 1 (Olfm1) is a conserved secreted glycoprotein expressed preferentially in developing and adult neuronal tissues. To better understand functions of Olfm1, we performed a shotgun proteomic analysis of proteins interacting with wild-type and mutated Olfm1 in the mouse postnatal day 1 total brain. Mutated Olfm1 has a deletion of 52 amino acids in the central part of the protein molecule that leaves the olfactomedin domain intact. Brain lysates were prepared and proteins interacting with Olfm1 were precipitated using affinity-purified antibodies against mouse Olfm1. Mass spectrometry analyses of the isolated complexes identified 105 candidate Olfm1-interacting proteins. Thirty one of these proteins interacted only with wild-type but not with mutant Olfm1. One of the proteins interacting with wild-type but not mutated Olfm1 is GluA2. GluA2 is a pore-forming subunit of AMPA-type glutamate receptors that are responsible for a variety of processes in the mammalian brain including fast excitatory neurotransmission, postsynaptic plasticity, or synapse development. GluA2 is also highly expressed in the retinal ganglion cells where it plays a role in a retina-specific form of AMPA receptor plasticity. Functional significance of Olfm1 interaction with AMPA receptor and other identified proteins is now being tested experimentally. The molecular changes in the brain of mice expressing mutated Olfm1were also analyzed by a high throughput RNA sequencing. The entire transcriptosomes from wild-type and Olfm1 mutant postnatal day 7 olfactory bulbs were analyzed. Among mRNAs sequenced, about 700 transcripts showed reduced abundance in Olfm1 mutant olfactory bulbs compared with wild-type samples. Analysis of transcripts with a reduced abundance suggested changes in synapses and cation channels in Olfm1 mutant olfactory bulbs compared with wild-type samples. We continued functional characterization of Olfm1 mutant line, Olfm2 and Olfm3 knockout lines, as well as lines deficient in different combinations of Olfm1-3 proteins. Retinal functions of Olfm1 mutant adult mice were analyzed by electroretinography (ERG) and visual evoked potential (VEP). There was no difference in ERG between wild-type and Olfm1 mutant lines. In VEP, although the amplitude of response showed no significant difference, the latency of response was significantly delayed in Olfm1 mutants compared to wild-type littermates. The number of retinal ganglion cells was counted in whole mount retina from 6-10 month old wild-type and Olfm1 mutant mice. A significant loss 25-75% of retinal ganglion cells was detected in Olfm1 mutant retina compared with wild-type littermates. In addition, Olfm1 mutant mice showed 25% decrease in a total optic nerve cross-sectional area compared with wild-type littermates. Olfm1 mutant mice showed behavioral abnormalities consistent with abnormal anxiety. Olfm1 mutant mice had reduced olfactory activity while the coordinated motor activity was normal. Although Olfm2 knockout mice shows no major phenotypical defects, analysis of the optic nerve by electron microscopy showed changes in the axonal organization and compactness of myelin sheath. A shift in the axon size was observed with knockout mice showing greater numbers of axons with > 1 microm2 area. A significant decrease in the VEP amplitude and slight decrease in latency was observed in Olfm2 knockout mice compared with wild type littermates. Our previous study has shown that Olfm1, 2 and 3 are homologous to each other, share similar expression patterns and interact with each other. Mutation in one of these genes inhibits the secretion of the others. Based on these results, we hypothesized that Olfm1, 2 and 3 may perform similar functions and might compensate for each other. In order to address this question, we generated triple knockout mice (Olfm1 mutant/Olfm2 knockout/Olfm3 knockout) and various combinations of double knockout mice. Most of the triple knockout mice die at early postnatal stages due to abnormal feeding behavior as the triple knockout pups lack a milk spot. The abnormal feeding behavior could be, at least partially, attributed to defects in the olfaction/ olfactory system, since the Olfm1 mutant and Olfm2 knockout mice and surviving double and triple knockout mice showed defects in olfaction as judged by an olfactory sensitivity test. In addition to olfaction deficiency, surviving double and triple knockout mice also showed severe defects in several behavioral tests. Some of the behavioral problems may be explained by changes in the AMPA receptor activity. We also used zebrafish to study functions of olfactomedin domain-containing proteins and regulation of their promoters. We have identified the upstream regulatory region of the zebrafish olfm2 gene and generated transgenic (F1) Olfm2 1 kb promoter fish for subsequent studies of its developmental and spatial regulation. We showed that 137 bp-5 kb olfm2 promoter fragments provided tissue-specific expression of (monomeric eGFP) mEGFP as early as 20 hours post fertilization (hpf). By 48 hpf, the expression reached the maximum level and was detected in the olfactory bulb, eye, optic nerve, optic tectum, telencephalon, anterior commissure, midbrain , hindbrain, branchiomotor neurons, spinal cord and other regions of anterior nervous system. In the eye, the expression was detected in the retinal ganglion cells as well as in the inner and outer nuclear layers. Binding sites for various transcription factors associated with neuronal proliferation, migration and differentiation were identified in the minimal promoter region. Mutations of two transcription factor binding sites, CCAAT box and NeuroD, reduced activity of olfm2 promoter.